Nitrite and nitrate in meat processing: Functions and alternatives

Meat and meat products are important foods in the human diet, but there are concerns about their quality and safety. The discovery of carcinogenic and genotoxic N-nitroso compounds (NOCs) in processed meat products has had serious negative impacts on the meat industry. In order to clarify the relationship between the use of nitrite or nitrate and the safety of meat or meat products, we reviewed NOCs in meat and meat products, the origin and safety implications of NOCs, effects of nitrite and nitrate on meat quality, national regulations, recent publications concerning the using of nitrite and nitrate in meat or meat products, and reduction methods. By comparing and analyzing references, (1) we found antioxidant, flavor improvement and shelf-life extension effects were recently proposed functions of nitrite and nitrate on meat quality, (2) the multiple functions of nitrite and nitrate in meat and meat products couldn't be fully replaced by other food additives at present, (3) we observed that the residual nitrite in raw meat and fried meat products was not well monitored, (4) alternative additives seem to be the most successful methods of replacing nitrite in meat processing, currently. The health risks of consuming processed meat products should be further evaluated, and more effective methods or additives for replacing nitrite or nitrate are needed.


Introduction
Meat and meat products are important sources of energy and nutrients for humans. The important position of meat and meat products in human foods is shown by the long history of their consumption, which can be traced back to about 3 million years ago (Chris, 2020;Guo et al., 2005;McKenna, 2019). Since mankind's earliest days, animal meat has been one of our species' major dietary materials, enabling primitive man to sustain a living. However, fresh meat is not stable to be stored for a long term. It will deteriorate within several days. This problem was solved by primitive man about 1.5 million years ago, with the application of fire to meat processing. Grilled meat from animals killed by forest fires tasted better than fresh meat. Therefore, they experimented with fire for cooking meat, discovering that roasted or smoked fresh meat was more stable for storage (Zeng, 2007). Roasting and smoking thus became the earliest preservation methods for meat and meat products. Possibly because of the effectiveness of smoking for preserving meat, and the habit of eating meat with a smoky flavor, roasted or smoked meat products are still popular and widely consumed globally.
The actual reason for the current use of nitrate and nitrite salts in cured meat products is related to the ancient salting practices for meat preservation. Nitrate and nitrite were contaminant in salts used in salted meats. The advances in science allowed the discovery of nitrate (NO 3 − ) first, and then nitrite (NO 2 − ), as actual ingredients involved in cured meat products preservation (Bedale et al., 2016). NO 2 − salts and NO 3 − salts, such as those of sodium and potassium, are typical food preservatives used in meat processing. Both of them can inhibit the growth of microorganisms, delay the onset of rancidity, produce cured meat flavor or smell, and stabilize the meat's red color (Ferysiuk and Wojciak, 2020). The efficiency of nitrite and nitrate makes them indispensable additives for meat and meat products, especially for cured meat products (Ferysiuk and Wojciak, 2020). However, the discovery of N-nitroso compounds (NOCs) in the 1950s raised concerns about the safety of using nitrite or nitrate in meat processing (Bedale et al., 2016), which seriously threatened the healthy development of the global meat industry. In addition, with widely acceptance of the Clean label movement, having fewer ingredients become mainstream in food processing (Asioli et al., 2017). In order to clarify the relationship between the use of nitrite or nitrate and the safety of meat or meat products, this review focuses on NOCs in meat and meat products, the origin and safety implications of NOCs, effects of nitrite and nitrate on meat quality, national regulations, recent publications concerning the using of nitrite and nitrate in meat or meat products, and reduction methods.

NOCS in meat and meat products
NOCs mainly consist of N-nitrosamines (RR'NNO) and N-nitrosamides (RN(NO)COR'). According to Dietrich et al. (2005) and Honikel (2008b), the proposed mechanism for the conversion of nitrite and nitrate to NOCs is shown in Fig. 1. RR'NNO and RN(NO)COR' are formed by the nitrosation of amines and amides, respectively. In vitro, thiocyanates naturally produced in foods accelerate the nitrosation of amines, and organic acid food additives catalyze the nitrosation of amides. Some bacteria with nitrate-reducing or nitrosating activity can also promote the formation of NOCs, but the production of NOCs can be inhibited by adding vitamins C and E (Dietrich et al., 2005), the other methods of reducing the production of NOCs were enumerated at section 7 of this review.
A list of recently identified NOCs in meat and meat products is shown in Table 1. There are 14 volatile and 11 non-volatile types of NOC. Among the N-nitrosamines reported most frequently in meat products, N-Nitrosodimethylamine (NDMA), and N-nitrosomethylethylamine (NDEA) are considered the most dangerous in terms of carcinogenicity and genotoxicity (De Mey et al., 2017;Ferysiuk and Wojciak, 2020). However, recent studies have identified N-nitroso-diisopropylamine (NDIPA) in sausages and ham using a new gas chromatography-tandem mass spectrometry method . According to NDIPA's safety description, it is a group 1B carcinogenic chemical, and more toxic than NDMA and NDEA. This discovery suggests that there might be other undetected toxic NOCs in meat or meat products, and therefore, developing new methods to identify the compounds is very important for the timely discovery and monitoring of NOCs in meat or meat products.

The origin and safety implications of NOCS
The addition of nitrite to meat or meat products may induce the formation of NOCs (Ferysiuk and Wojciak, 2020). The World Cancer Research Fund stated that eating red or processed meat was likely to increase the risk of colorectal cancer (CRC) in 2007 (Rajkovic et al., 2017). Given the higher cancer risk associated with eating red and processed meat (Crowe et al., 2019), the IARC classified all types of mammalian muscle meat, such as beef, veal, pork, lamb, mutton, horse, and goat, as probably carcinogenic (Group 2A), and processed meat (treated with salting, curing, fermentation, smoking, or other processes to enhance flavor or improve preservation) as carcinogenic to humans (Group 1) (IARC, 2018).
Exposure to nitrite or nitrate may occur via many sources besides red meat and its related products. Table 2 indicates that consumption of vegetables is responsible for approximately 80% of nitrite exposure and 85% of nitrate exposure, while the consumption of meat and meat products accounts for only about 5% of nitrite exposure (Ferysiuk and Wojciak, 2020). Although these exposure ratios may be only approximate, they demonstrate that vegetables, nitrate reduction, and water are the three major sources of nitrite exposure in humans. Further investigation is needed into the contributions of consuming meat or meat products treated with nitrite to cancer risks in humans. In order to promote the healthy development of the meat industry and ensure human health, the purpose of this review is to evaluate the recent developments and current knowledge concerning nitrite and nitrate in meat processing and alternative methods to reduce the residual amount of nitrite and nitrate in meat or meat products.

Color formation effect
Nitrite or nitrate can produce a pinkish-red color via interaction with the myoglobin in muscle. In addressing the food safety concerns, most researchers have, in recent years, focused on finding replacement color formation ingredients containing natural nitrite or nitrate, such as vegetable extracts and bacteria. Jarulertwattana et al. (2021) found that 4 × 10 − 6 g/g of nitrite was suitable for chicken meat marination when it was used with ginger paste, whereas more than 4 × 10 − 6 g/g of nitrite will produce pink color defect in the drumsticks. Zhu et al. (2020) used Lactobacillus plantarum to partially replace standard nitrite in pork sausages, finding that 50 mg/kg NaNO 2 combined with 7 log CFU/g L. plantarum produced similar effects to 100 mg/kg NaNO 2 alone. Ozaki et al. (2020) added radish powder (0.5% or 1.0%) and oregano essential oil (100 mg/kg) to fermented cooked pork and beef sausage, and found that both treatments could improve the red color of these products. Higuero et al. (2020) found that 75 mg/kg nitrate or 37.5 mg/kg nitrite produced a similar color to that seen following 150 mg/kg nitrate or 150 mg/kg nitrite treatment. This research suggests that, in terms of red color formation, there is an optimal amount of nitrite or nitrate. Therefore, appropriate process optimization may be an advisable method for controlling the amount of nitrite or nitrate added. Typical process optimization methods are orthogonal experiment design and response surface experimental design (Zhang et al., 2012). The latter, in particular, has been successfully used in the optimization of processes involving meat or other food products (Zhang et al., 2014(Zhang et al., , 2015Zhao et al., 2017a). Recently, Kim and Chin (2021) evaluated the effects of paprika oleoresin, sunflower seed oil, and sodium nitrite on the storage quality of emulsified pork sausage, and the results indicated that this combination, given as 7.5 × 10 − 5 NaNO 2 + 0.1% paprika oleoresin solution (1% paprika oleoresin +99% sunflower seed oil or 5% paprika oleoresin +95% sunflower seed oil) could increase redness.

Antimicrobial properties
The antimicrobial effects of nitrate and nitrite are considered very important in meat and meat products. The minimum concentration of nitrite that inhibits the outgrowth of Clostridium botulinum is 4 × 10 − 5 -8 × 10 − 5 g/g (Rivera et al., 2019). Patarata et al. (2020) investigated the effects of red wine and garlic on the behavior of C. sporogenes (used as a substitute for C. botulinum) and Salmonella in a dry-cured chouriço sausage, and found that red wine (7.5%) and red wine + garlic (7.5% + 1%, respectively) could destroy Clostridium sporogenes and Salmonella during the processing of chouriço. Khatib et al. (2020) used hop components to replace nitrite in the preparation of cooked beef sausage by means of lupulon-xanthohumol-loaded nanoliposomes (XLN) used in the production process; the results indicated that 1.5 × 10 − 4 XLN and 3 × 10 − 5 nitrite could produce bacteriostasis during 30 days of storage at 4 • C. Radish powder and oregano essential oil can produce nitrite in fermented cooked pork and beef sausages, thus inhibiting mesophilic bacteria (Ozaki et al., 2020). Kim and Chin (2021) found that the addition of paprika oleoresin, sunflower seed oil, and sodium nitrite (7.5 × 10 − 5 NaNO 2 + 0.1% paprika oleoresin solution [1% paprika oleoresin +99% sunflower seed oil or 5% paprika oleoresin +95% sunflower seed oil]) could decrease the total plate counts of emulsified pork sausage samples. Vegetable extracts alone or in combination with other ingredients thus show promising abilities to control microorganisms in meat or meat products, but the specific chemicals that result in the antimicrobial effect are not well understood. Therefore, more investigations should be performed to identify the underlying processes responsible for the antimicrobial effect of vegetable extracts on meat or meat products.

Flavor improvement
The effect of microbial starters on the curing flavor of meat products has become a hot research topic. Perea-Sanz et al. (2020) investigated the effect of Debaryomyces hansenii inoculation on the aroma chemicals of dry fermented sausage and found that D. hansenii could induce the generation of potent aroma compounds such as ethyl ester and 3-methylbutanal. TIAN et al. (2020b) used Lactobacillus fermentum RC4 to decrease the nitrite level in salted meat, and the results indicated 73 volatile substances were identified in the fermented group, while there were 67 volatile substances in the control. Zhao (2020) investigated the effects of lactobacillus helveticus TR1-1-3 and ZF22 on the flavor chemicals of mutton sausages, and found that 1-pentene-3-ol, 1-octene-3-ol, and 3-hydroxy-2-butanone were typical flavor chemicals in fermented sausages. Huang et al. (2021) investigated the effects of Lactobacillus starter culture (Lactobacillus fermentum RC4, L. plantarum B6) on the volatile flavor elements in cured meat. The results indicated the key volatile components included D-limonene and nonanal, (E,E)-2, 4-decadienal, 1-octene-3-ol, and anethole. Nitrite can inhibit the growth of bacteria like Clostridium botulinum, Bacillus cereus, Staphylococcus aureus, Clostridium perfringens, and others (Dong and Tu, 2006). This effect of nitrite can assist some fermentative bacteria in cured meat products to produce fermented flavor ingredient by the metabolism of themselves or the hydrolysis of proteases and lipases in them Wang et al., 2021). Therefore, nitrite may indirectly affect development of cured meat flavor via its impact on the activity of microorganisms and endogenous enzymes.

Antioxidant effect
The NO engendered by nitrite can competitively deplete oxygen by self-oxidation, bind to the iron ion in hemoglobin and thus prevent its oxidization, and destroy the radical chain reactions of lipid oxidations (Jo et al., 2020). These indirect effects of nitrite may explain the antioxidant effects of nitrite. Ji et al. (2020b) found that sodium nitrite can effectively inhibit lipid oxidation at 1 × 10 − 4 in mutton marinating process. Khatib et al. (2020) used L-X-NL (lupulon-xanthohumol loaded nanoliposome) to replace nitrite in the preparation of cooked beef sausage, and found that these nanoliposomes could prevent lipid oxidation. Ma et al. (2021) compared the antioxidant effects of phosphorylated nitrosohemoglobin (PNHb) and sodium nitrite on emulsified sausage; the results indicated that the thiobarbituric acid values of the PNHb group and NaNO 2 group were 0.62 mg/kg and 0.67 mg/kg, respectively, suggesting that PNHb has a stronger antioxidant effect than NaNO 2 . Despite the results, nitrite is still widely accepted as a color formation and antiseptic agent in most countries.

Shelf-life extension
The improvement in shelf life is actually a combination of many factors delaying the loss of quality, including the hygiene of raw meat, processing treatments, storage temperature and relative humidity, packaging method, etc (Singh et al., 2011). For nitrite and nitrate, they can extend the shelf-life of meat and meat products by inhibiting the outgrowth of pathogenic and spoilage bacteria (Rivera et al., 2019). Recently, Chatkitanan and Harnkarnsujarit (2020) combined thermoplastic starch, sodium nitrite, and low-density polyethylene to develop a starch-based, nitrite-containing film. They used it to vacuum-package pork, and the results indicated that the film not only increased the redness of pork by more than 30% during chilled storage, but also inhibited microbial growth and lipid oxidation. Khatib et al. (2020) found that the addition of XLN to cooked beef sausage could extend its storage life via bacteriostasis and prevent fat oxidation during 30 days of storage at 4 • C.

Regulation of nitrite and nitrate use
The multiple functions of nitrite and nitrate in meat and meat products cannot be fully replaced by other food additives at present (Karwowska and Kononiuk, 2020). Complete restriction of their use in this context may not be acceptable to producers and consumers. In order to regulate the application of nitrite and nitrate in meat or meat products, many countries have established directives and regulations (Table 3) (Gassara et al., 2016).
Germany is the fourth largest meat producer in the world (Fig. 2), and cured meat products are popular there. It was the first to propose limits to the addition of nitrite to meat and meat products, restricting the nitrite content in curing salt to between 0.5% and 0.6% (Ferysiuk and Wojciak, 2020). The United Nations only limits residual nitrite levels according to the category of meat product (Table 3). The United States is  (Chan, 2011;Gassara et al., 2016;Leth et al., 2008) Water Drinking water, beverage, aquatic products, liquid condiments, agriculture products fertilized with natural organic wastes or overused fertilizers approximately 5-40% dietary nitrate intake, if the nitrate level in drinking-water is more than 50 mg L − 1 , it wiil be the main source of exposure to nitrates (Addiscott, 2005;Elias et al., 2020;Karwowska and Kononiuk, 2020) Meat and meat products Fresh meat, chilled meat, freezing meat, cured meat products, sauced and stewed meat, roasting meat burning smoke, dried meat, fried meat products, sausage meat products, prepared meat products, ham sausage, others approximately 5% nitrite exposure, it depends the category of meat and meat products (Colla et al., 2018;Ferysiuk and Wojciak, 2020   Maximum residual sodium nitrite level (expressed as NaNO 2 ) is 5 × 10 − 5 -1.75 × 10 − 4 Maximum added sodium nitrate amount (expressed as NaNO 2 ) during manufacturing ≤3 × 10 − 4 ; Residual sodium nitrate amounts (expressed as NaNO 2 ) is ≤ 5 × 10 − 5 (some without added) Wei et al. (2009)

China
Cured meat products (such as bacon, cured meat, salted duck, Chinese ham and sausage) Maximum addition amount (sodium (potassium) nitrite) ≤ 0.5 g/kg; Based on sodium nitrite, the residue is ≤ 3 × 10 − 5 Maximum addition amount (sodium (potassium) nitrate) ≤ 0.5 g/kg; Based on sodium (potassium) nitrite, the residue is ≤ 3 × 10 − 5 National Health and Family Planning Commission (2015) sauced and stewed meat products Maximum addition amount (sodium (potassium) nitrite) ≤ 0.5 g/kg; Based on sodium nitrite, the residue is ≤ 3 × 10 − 5 Maximum addition amount (sodium (potassium) nitrate) ≤ 0.5 g/kg; Based on sodium (potassium) nitrite, the residue is ≤ 3 × 10 − 5 National Health and Family Planning Commission (2015) Smoked and roasted meat products Maximum addition amount (sodium (potassium) nitrite) ≤ 0.5 g/kg; Based on sodium nitrite, the residue is ≤ 3 × 10 − 5 Maximum addition amount (sodium (potassium) nitrate) ≤ 0.5 g/kg; Based on sodium (potassium) nitrite, the residue is ≤ 3 × 10 − 5 National Health and Family Planning Commission (2015) Fried meat products Maximum addition amount (sodium (potassium) nitrite) ≤ 0.5 g/kg; Based on sodium nitrite, the residue is ≤ 3 × 10 − 5 Maximum addition amount (sodium (potassium) nitrate) ≤ 0.5 g/kg; Based on sodium (potassium) nitrite, the residue is ≤ 3 × 10 − 5 National Health and Family Planning Commission (2015) Western style ham (smoked and roasted, smoked, stewed) Maximum addition amount (sodium (potassium) nitrite) ≤ 0.5 g/kg; Based on sodium nitrite, the residue is ≤ 7 × 10 − 5 Maximum addition amount (sodium (potassium) nitrate) ≤ 0.5 g/kg; Based on sodium (potassium) nitrite, the residue is ≤ 3 × 10 − 5 National Health and Family Planning Commission (2015) Sausage meat products Maximum addition amount (sodium (potassium) nitrite) ≤ 0.5 g/kg; Based on sodium nitrite, the residue is ≤ 3 × 10 − 5 Maximum addition amount (sodium (potassium) nitrate) ≤ 0.5 g/kg; Based on sodium (potassium) nitrite, the residue is ≤ 3 × 10 − 5 National Health and Family Planning Commission (2015) Fermented meat products Maximum addition amount (sodium (potassium) nitrite) ≤ 0.5 g/kg; Based on sodium nitrite, the residue is ≤ 3 × 10 − 5 Maximum addition amount (sodium (potassium) nitrate) ≤ 0.5 g/kg; Based on sodium (potassium) nitrite, the residue is ≤ 3 × 10 − 5 National Health and Family Planning Commission (2015) Canned meat products Maximum addition amount (sodium (potassium) nitrite) ≤ 0.5 g/kg; Based on sodium nitrite, the residue is ≤ 5 × 10 − 5 National Health and Family Planning Commission (2015) Canada Cured meat and meat by-products (except bacon) the second largest meat producer globally (Fig. 2), and its cured meat and meat products are frequently consumed as hotdogs or other convenience foods (Table 3). The maximum nitrite level for pumped and/or massaged bacon (the ingoing level of sodium nitrite ≤120 (or 148 ppm potassium nitrite) ppm) and other meat products were restrictedly limited (Table 3). More specific restrictions have been imposed by the European Union and European countries (Table 3). They have restricted the levels of nitrite and nitrate in treatments and the residue levels in different meat products (Table 3). China is the world's largest meat producer (Fig. 2), cured meat products being widely-consumed traditional foods. To regulate the application of nitrite and nitrate in meat or meat products, similar limits have been proposed within Chinese additive standards (Table 3) (National Health and Family Planning Commission, 2015). Canada, Korea, Japan and other countries are all restricting input and residue levels of nitrite and nitrate in meat or meat products (Table 3).
Restricting input and residue levels is beneficial, in that it prevents the overuse of nitrite and nitrate in meat or meat products, but it is also essential to ensure that directives or regulations are implemented appropriately. Although most countries have established food inspection agencies to supervise the use of nitrite and nitrate in meat or meat products, there will be some omissions. Abd Hamid et al. (2020) investigated the nitrite intake of school children in Brunei Darussalam, and found that more than 20% of children exceeded the Acceptable Daily Intake (ADI) of nitrite (<0.07 mg/kg b. w/day). Therefore, frequent market sampling inspections are necessary to ensure the implementation of directives or regulations concerning nitrite or nitrate levels.

Nitrite and nitrate in meat and meat products
Meat production is an important index within global agriculture. The top ten meat-producing countries from 2015 to 2019, according to the Food and Agriculture Organization of the United Nations (FAO), are shown in Fig. 2. China and the USA are the two largest meat producers, jointly accounting for more than 60% of total world output. As such, their classification systems and data for meat products are the most detailed, and are reviewed below. These countries categorize meat and meat products into 11 types.
Two categories cover meat: raw meat and cooked meat; the remaining nine categories cover processed meat products (Table 4). Based on the categories listed in Table 4, the residual nitrite and nitrate in sausages (12) and ham (7) are the most frequently reported, while other types of meat products, in decreasing order of frequency, are roasted and burning smoked meat products (5), raw meat (4), cured meat products (4), dried meat products (2), other meat products (2), and sauced and stewed meat products (1). Few reports have been published concerning the residual nitrate or nitrite in fried meat products. The residual levels of nitrite and nitrate in sausage were 0-1.39 × 10 − 4 and 1.88 × 10 − 4 or less, respectively (Table 4). The residual levels of nitrite and nitrate in ham were 5.76 × 10 − 5 and 1.94 × 10 − 4 or less, respectively (Table 4). According to the European Commission's former Scientific Committee for Food and the Joint FAO/WHO Expert Committee on Food Additives, the ADIs for nitrite and nitrate are important for the health of consumers (Karwowska and Kononiuk, 2020). The recommended ADI for nitrite is less than 0.06 mg/kg bw/day; that for nitrate is 3.7 mg/kg bw/day (Santamaria, 2006). Taking the maximum amounts of nitrite (1.39 × 10 − 4 ) and nitrate (1.88 × 10 − 4 ) in sausage as examples, if an adult weighing 60 kg consumes 60 g of sausage, their nitrate intake will be below its ADI (0.188 mg/kg bw/day <3.7 mg/kg bw/day), but their nitrite intake will be above the daily acceptable level (0.139 mg/kg bw/day >0.06 mg/kg bw/day). Therefore, stricter application of regulatory restrictions and more frequent sampling surveys are required in countries where the ADIs for nitrite and nitrate are greater than 0.06 and 3.7 mg/kg bw/day, respectively.
To avoid indiscriminate use of nitrite and nitrate in meat or meat products, the scope for their addition is rigorously restricted in most countries. However, the data in Table 4 indicate that nitrite or nitrate have been added to raw meat materials, such as minced meat, beef, lamb, chicken meat, and fish. As ingredient or hazardous compound levels are not monitored by regulation in raw meat materials in some countries, and considering that nitrite overuse in processed meat products has occurred (Abd Hamid et al., 2020;Neri and Patalinghug, 2021), the application of nitrite or nitrate in raw meat materials may be a supervisory blind spot that increases the risk of overuse. Therefore, it is vital to strengthen the sampling regime for meat and meat products, especially raw meat where it is not currently included.
Fried meat products comprise one of the most widely consumed meat categories, but there are not many reports on their residual levels of nitrite or nitrate (Table 4). This may be because fried meat products are

Fig. 2.
Top 10 countries in meat production quantity.  prepared meat products, which are readily consumed mixed with other foods, and usually eaten immediately after cooking. These features make them popular among adults or children and they are widely consumed in snack bars and homes. An investigation of nitrite intake in school children in Brunei indicated that more than 20% exceeded their nitrite ADI (0.06 mg/kg bw/day) by eating cured meat products (Abd Hamid et al., 2020). Therefore, in order to evaluate the safety hazards of eating fried meat products, more surveys of the residual nitrite or nitrate in fried meat products should be conducted, especially in products consumed by infants or children.

Alternative additives
Following the classification of nitrite-cured meat as a Group 1 carcinogen by IARC (IARC, 2018), many researchers have tried to find alternative additives to replace nitrite or nitrate. Vitamins, vegetable extracts, spices, herbs, and fruits have all been investigated as alternatives (Flores and Toldra, 2021;Gassara et al., 2016), as these materials or their ingredients had shown antioxidant and/or bacteriostatic properties, preventing the formation of nitrosamines. Bamboo leaf extracts were found to prevent nitrite from transforming into N-nitrosamines by Pan et al. (2019), who added it to pork ham; the addition of 0.2% bamboo leaf extract could effectively stop the transformation of nitrite to N-nitrosamines. Wang et al. (2020) used rose extract to substitute for nitrite in the preparation of semi-dried fermented sausage, the resulting sausage quality was significantly improved over that of the sausage with 150 mg/kg sodium nitrite; the optimal combination was 10% rose extract and 80 mg/kg sodium nitrite. Khatib et al. (2020) prepared XLN and used them to replace nitrite in the preparation of cooked beef sausage, finding that this treatment can partially replace nitrite (50%) in the preparation of cooked beef sausage without impairing its sensory quality. Ozaki et al. (2020) added radish powder (0.5% or 1.0%) and oregano essential oil (100 mg/kg) to fermented cooked pork and beef sausages, and found that this mixture could improve their color and inhibit mesophilic bacteria, but it could not prevent lipid oxidation. Alirezalu et al. (2020) mixed plant extracts (stinging nettle, olive leaves, and green tea) with nisin or nisin nanoparticles, and compared their effects on color, fat oxidation, and the microorganism content of frankfurter sausages, finding that the mixture of plant extracts and nisin nanoparticles could prevent fat oxidation and inhibit the increase of bacteria, molds, and yeasts. They considered that the use of 2 × 10 − 4 g/g nisin nanoparticles and 5 × 10 − 4 g/g of mixed plant extract could enable production of nitrite-free frankfurter sausages of good quality with a stable shelf life. Recently, the addition of 25% guava epicarp flour extract in the production of frankfurters (Velasco-Arango et al., 2021), 0.2% Flos Sophorae and 1% chili pepper in Chinese sausages (Tang et al., 2021), 0.3% white kimchi powder and 0.5% lemon extract powder in naturally cured sausages (Bae et al., 2021), extracts of wild thyme by-product and potassium bixinate were all found effective to replace nitrite (Sojic et al., 2022). Other extracts from cruciferous vegetables as sources of nitrate in meat were reviewed by (Munekata et al., 2021).

Microbial degradation
Beyond the addition of vegetable juice or extracts to reduce the residual nitrite in meat or meat products, some microbes also showed nitrite reduction and conversion effects (Table 5). Microbial enzymes can convert metmyoglobin to NO-Mb, thereby making the cured meat pink. Huang et al. (2019) added Lactobacillus fermentum RC4 and L. plantarum B6, without any nitrite, during preparation of cured meat, producing a bright color and low nitrite content. Huang et al. (2020b) adopted three staphylococcal species (S. vitulinus, S. carnosus, and S. equorum) to induce the production of NO-Mb through nitric oxide synthase catalysis, which, in all three species, increased the a* value of the dry sausage model. Zhang et al. (2020b) inoculated Lactobacillus curvatus LAB26 and Pediococcus pentosaceus SWU73571, isolated from the sour meat of the Dong minority in China, into sour meat. Both bacteria decreased the nitrite content of sour meat and increased the a* value significantly. Luo et al. (2020) added Lactobacillus fermentum AS1.1880 into pork batters and confirmed that the species increased their redness by producing nitric oxide synthase. Tian et al. (2020a) found that Lactobacillus fermentum RC4 significantly reduced the nitrite  (Alahakoon et al., 2015;MoLler et al., 2003;Xue et al., 2007;Zhang et al., 2007)  Bacon Liu et al. (2021) content of salted meat. They combined Lactobacillus fermentum RC4 and L. plantarum B6 with beet red, Monascus red, and nisin to produce Chinese bacon, and found that the combined agents not only improved the cured meat quality, but also decreased the nitrite content. Liu et al. (2021) used the commercial starter PRO-MIX5 (Staphylococcus xylose, Lactobacillus sakei, Lactobacillus plantarum) to prepare bacon and found that the starter resulted in significantly lower NDMA and total N-nitrosamine levels.

Cooking method
Nitrite is easily oxidized in high-temperature aerobic environments, such as cooking. Therefore, cooking can decrease the nitrite content of meat or meat products. Nitrate reduction and nitrite accumulation were observed in the uncooked sausage models, but Waga et al. (2017) found that nitrate inhibits nitrite auto-decomposition in pork ham cooked in a water bath at 75 • C. Asioli et al. (2017) investigated the effects of dry-frying temperatures (100, 150, 200, and 250 • C) on the residual nitrite and N-nitrosamine levels in smoked bacon, and found that the residual nitrite content initially increased (from unheated control to 150 • C) and then sharply decreased between 150 • C and 250 • C. Sallan et al. (2020) investigated the effects of cooking extent on volatile nitrosamine formation in the heat-treated semi-dry fermented sausage sucuk, and found that higher temperature and higher heat intensity induced more nitrite controverted into nitrosamines, the content of nitrite and cooking levels influenced the content of N-nitrosopiperidine (NPIP) more than other nitrosamines, well done and very well done cooking levels combining with higher nitrite contents (100 and 150 mg/kg) resulted in significant increases in NPIP content. Recent investigations into the effects of cooking in this context recommend it for the reduction of residual nitrite in meat and meat products.

Irradiation
Food irradiation includes treatment with gamma rays, X-rays, or electron beams. Among these methods, gamma irradiation has the highest potential. However, its development and commercialization have been hampered by unfavorable public perceptions. The endorsement of food irradiation by many international food and health organizations has counteracted this perception to some degree; thereby increasing consumers' confidence and raising food industry interest. Gamma irradiation is effective in reducing the residual nitrite and Nnitrosamines in meat products. A model sausage that was irradiated with γ-irradiation found that high dose irradiation (>10 kGy) could decrease the residual nitrite levels significantly (Ahn et al., 2002). A dose of 5 kGy γ-irradiation significantly decreased residual nitrite in Chinese Rugao ham before ripening (Wei et al., 2009). In recent years, limited investigations on effect of irradiation on reduction of the residual nitrite were reported.

Plasma-treated water
The interaction of plasma with water can result in the generation of nitrate and nitrite, as well as reactive oxygen species Moutiq et al., 2020). Therefore, plasma-treated water can be used as a substitute for nitrite or nitrate. Moutiq et al. (2020) used atmospheric cold plasma to treat chicken breast meat and found that levels of natural microflora were approximately 2 log CFU/g lower following treatment with 100 kV atmospheric cold plasma for 5 min, and that the shelf life of the chicken breast was extended by sterilization of the mesophiles, psychrotrophs, and Enterobacteriaceae in the meat. Dong and Tu (2006) used atmospheric nonthermal plasma to treat roasted lamb, and found that 45 min plasma treatment resulted in 30% lower residual nitrite compared with the lamb treated with added nitrite, while the overall sensory scores were similar in both groups.

Other technologies
As the food industry develops, clean labels are widely preferred and demanded by consumers. Therefore, natural food additives, nondestructive technologies, and minimally harmful processing technologies will be developed to reduce or replace the addition of nitrite or nitrate (Ferysiuk and Wojciak, 2020;Fraqueza et al., 2018;Ranasinghe, 2018;Zhang et al., 2016;Zhang et al., 2020a). In terms of naturally sourced additives, there are more than 2000 edible vegetables, extracts of most of which remain to be evaluated for nitrite reduction. Among non-destructive technologies, ohmic heating and its combination with other non-destructive technologies have not yet been investigated. Minimally harmful processing technologies with potential include bio-preservation methods, bacteriocinogenic cultures, and genetically modified fermentation strains, all of which need to be investigated.
The advantages and disadvantages of ten alternative methods are listed in Table 6. Among them, the use of alternative additives, bacterial fermentation, improved packaging, and high hydrostatic pressure methods have all been frequently investigated in recent years; the replacement of nitrite and nitrate with vegetable extracts has been industrialized, and some natural additives have been commercialized. Meat quality improvement methods have been evaluated and tried in meat production, but the resulting products are expensive and are not accepted by most consumers. Cooking and storage condition methods are used in food processing and storage. Irradiation, plasma-treated water, and UV light methods are emerging technologies, and their main function is to sterilize spoilage bacteria and pathogens in meat or meat products. However, their effects on color, flavor, and oxidation of meat or meat products remain to be further investigated.

Conclusions
Nitrite is a vital additive in meat and meat products. To date, the multiple functions of nitrite in meat processing cannot be completely replaced by other additives. To ensure that the use of nitrite or its oxide nitrate is safe for humans, regulations and directives have been adopted by various countries and regions, and 6 alternative methods of reducing or partially replacing nitrite in meat processing have been described in this review. Among these methods, alternative additives may be the most successful methods of replacing nitrite in meat processing, because there were many recently investigations focused on this direction. The analysis and comparison of recent investigations into nitrite and nitrate in meat and meat products have found that there are many unanswered questions that require further investigation. We suggest that the correlation between the risk of cancer and consumption of cured meat should be systematically researched. In particular, the main contributors to CRC among vegetables, water, red meat, and meat products should be clarified. The sampling survey by regulators should pay more attention to raw and fried meat products. Ohmic heating and other alternative methods or additives, which can improve the cleanliness of labels and are conducive to the production of high-quality meat or meat products, should be further studied and developed.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability
Data will be made available on request.

Table 6
Advantages and disadvantages of the alternative methods.

Alternative methods
Advantages Disadvantages

Meat quality improvement
Reduce or partially replace nitrite or nitrate, improve the flavor of meat Storage intolerance, high feeding cost, the meat price is high Alternative additives Reduce or partially replace nitrite or nitrate, enrich nutrients of meat products, improve the flavor of meat products Effect is not as good as adding nitrite, need a variety of additives, increased safety control risk,

Bacteria fermentation
Reduce or partially replace nitrite or nitrate, produce unique flavor, extend shelf life, improve product texture and protein absorption rate; reduce the pH and moisture content The production process is complex, there is the risk of harmful bacteria pollution, the production cost is high, some products tastes sour Cooking Reduce or partially replace nitrite or nitrate, saves cost, it can make the product produce good flavor, the cooked meat or meat products are convenient to eat It is mainly applicable to ready to eat products, long-term steaming will lead to the increase of nitrite content in sausage and cured meat, require heat energy Storage conditions Reduce the residual nitrite content, low operation cost, easy to implement, it is not easy to cause deterioration of meat or meat products Sensory quality of meat or meat products will be influenced, it takes a long time, prolonged exposure to oxygen can easily lead to fat or protein oxidation High hydrostatic pressure Reduce or partially replace nitrite or nitrate, it can kill microorganisms, reduce nutrient loss caused by heat treatment, it can be used to process food materials with new functional characteristics, it can be carried out at room temperature or even lower, the processing procedure is simple; there are no strict requirements on the size and shape of meat or meat products High equipment cost, not applicable to the processing of large meat products, the meat products should be vacuum packed before processing, ultra high pressure process requires intermittent operation, there is a risk of cross contamination between pressure medium and meat and meat products Irradiation Reduce or partially replace nitrite or nitrate, it can prevent and control foodborne pathogenic bacteria and spoilage bacteria and keep the quality of meat products Long time irradiation will produce peculiar smell, not suitable for raw meat and its products, it will cause the loss of nutrients to a certain extent, it is harmful to human body and requires special protection Plasma-treated water Reduce or partially replace nitrite or nitrate, fast sterilization speed, high sterilization efficiency, pollution-free, efficient and environmental protection It is suitable for sterilization of unpacked meat and meat products, for surface sterilization only, it damage protein structure

Package method
Reduce or partially replace nitrite or nitrate, It can maintain the quality, color, flavor and nutrition of meat and meat products, It can prolong the shelf life of meat and meat products It is not suitable for irregular bone meat and its products, vacuum packaging can lead to the accumulation of juice on the surface of meat products, packaging materials pollute the environment Illumination method Reduce or partially replace nitrite or nitrate, high sterilization efficiency and simple equipment, no toxic and harmful substances will be produced, it has a certain odor removal effect, the equipment covers a small area and is easy to maintain It is only applicable to the surface sterilization of meat and meat products, there is no continuous disinfection ability, there may be a problem of microbial photoreactivation, it cannot sterilize spores, cysts and viruses